The C2C12BRELuc reporter cell line [41 (link)] was generated by the Inman group and used in this study. In this cell line, the luciferase (Luc) reporter is under the control of the BMP response elements (BRE) of the Id1 gene. C2C12BRELuc were cultivated in culture medium (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, all from Biochrom AG, Berlin, Germany). C2C12BRELuc cells were passaged twice per week in a 175 or 225 cm2 cell culture flask. The cultivation media was supplemented with the antibiotic G418 (0.7 mg/mL) to select for the BREluc-positive C2C12BRELuc cells in accordance with previous studies [41 (link)].
C2C1BRELuc cells were sub-cultivated at a ratio of 2.5–5 × 104 cells per mL of culture medium. DMEM, antibiotics, FBS, and trypsin (Biochrom AG, Berlin, Germany) were used in cell cultivation and passaging. To ensure the viability and to calculate cell number, the metabolic activity of cells was calculated through Presto Blue® assay (Invitrogen by Life Technologies Co., Carlsbad, CA, USA). BMP2 was purchased from Osteogenetics (Würzburg, Germany). H89 was purchased from AbCam (Cambridge, UK). Deta NONOate, SNAP, YC-1, LY83583, LDN-193189, and IBMX were purchased from Cayman Chemical (Ann Arbor, MI, USA).
C2C1BRELuc cells were sub-cultivated at a ratio of 2.5–5 × 104 cells per mL of culture medium. DMEM, antibiotics, FBS, and trypsin (Biochrom AG, Berlin, Germany) were used in cell cultivation and passaging. To ensure the viability and to calculate cell number, the metabolic activity of cells was calculated through Presto Blue® assay (Invitrogen by Life Technologies Co., Carlsbad, CA, USA). BMP2 was purchased from Osteogenetics (Würzburg, Germany). H89 was purchased from AbCam (Cambridge, UK). Deta NONOate, SNAP, YC-1, LY83583, LDN-193189, and IBMX were purchased from Cayman Chemical (Ann Arbor, MI, USA).